We propose to continue a multidisciplinary project to understand the mechanisms by which aberrant RNA and DMAstructures trigger gene silencing in a model animal system, the nematode Caenorhabditis elegans. The specific aims of the proposed research are 1. Investigate the roles and mechanisms for trigger amplification in the C. elegans gene silencing machinery. 2. Investigate interconnections between gene silencing, genome defense, and development in C. elegans. 3. Identify additional structures in DNA and RNA that act as triggers for gene silencing. 4. Continue to develop and refine tools and assays for studies of genetic activity and silencing in C. elegans. Studies of gene silencing have been of value from numerous perspectives and we hope the results of our work to impact several diverse aspects of biomedical research. First, an understanding of gene silencing mechanisms allows us to better design systems for expressing specific genes in vivo. Such expression systems can provide significant advantages for applications such as gene therapy, analysis of mutant protein function, protein production, and general investigation of protein activity. Second, as we understand mechanisms of gene silencing, we acquire the ability to specifically and effectively silence genes within cells or in an organism, generating a significant toolkit for functional genomic research, and aiding in the development of tools for gene-based therapeutics. Third, because gene silencing mechanisms are indicative of a variety of cellular gene regulation mechanisms, work on gne silencing has provided valuable insights into normal gene regulation. Fourth, many gene silencing mechanisms reflect the response of the cell/organism to DNA or RNA that is viewed as foreign. This type of response forms the basis of mechanisms in place to resist "selfish DNA" (or selfish RNA) in the form of viruses and transposons. Studies of gene silencing can thus extend our understanding of natural mechanisms used to fight viral infection (and by extension allow those mechanisms to be more readily induced or effected when needed).